Avian blood may contain various disease agents, including viruses, bacteria, rickettsiae, protozoa, microfilariae, and rarely fungi. These organisms can be identified by microscopic examination of wet mounts, buffy coat, or blood smears or by appropriate culturing and molecular techniques. Microscopically, some are within blood cells (Plasmodium, Haemoproteus, Leucocytozoon, Isospora , Hepatozoon, Babesia, Aegyptianella), whereas others are free in the plasma (Trypanosoma, microfilariae, bacteria, spirochetes). None live exclusively in the blood; most are found in tissues but are present in blood during part of their life cycle. Some, such as microfilariae and Plasmodium, have numbers or stages of parasites that vary with time.
In such cases, examining multiple smears at intervals will increase the likelihood of obtaining a diagnosis. Seasonal variations in infection rates relate to the activity of arthropod vectors. Most bloodborne organisms are either uncommonly or not associated with clinical disease. Very young, weakened, or injured birds infected with hemoprotozoa may have higher mortality and slower recovery than uninfected birds. Examination for bloodborne organisms should be included in the clinical and diagnostic procedures for ill birds.
Thin blood smears should be made with blood directly from the bird, if possible. Anticoagulants, storage, and cooling of the blood can distort protozoal morphology and introduce artifacts. A Romanowsky-type stain that gives good polychromatic coloration (eg, Giemsa stain) should be used.
Bloodborne organisms in plasma or WBCs are concentrated in the buffy coat. Stained buffy coat smears are recommended to detect bacteria, spirochetes, and Leucocytozoon, Trypanosoma, or Isospora infections. Direct examination of the buffy coat by darkfield or phase contrast microscopy is an excellent technique to identify low numbers of motile organisms such as spirochetes and microfilariae. The buffy coat and all of the plasma should be expressed onto a glass slide and covered with a coverglass, which is depressed slightly to spread the buffy coat. The buffy coat/plasma interface should be examined with darkfield or reduced light microscopy to detect motile organisms.
To make a diagnosis of infection with an intracellular blood protozoan on a thin blood film, it first should be determined that the organisms in question are neither normal structures nor artifacts. The following should then be determined: the host cell and whether it is normal or deformed beyond identification, whether pigment granules (hemozoin) are present or absent, and whether merogony is occurring ( see Table: Characteristics of Protozoa Encountered in Avian Blood Characteristics of Protozoa Encountered in Avian Blood ). Identification of an organism beyond genus is difficult and usually unnecessary for clinical purposes.
Organ cytology (impression smears) and blood smears are useful adjuncts to histopathology for postmortem examination.
Serologic and molecular diagnostic methods have been developed for avian hemoparasites. Molecular methods are very sensitive and can detect infection when parasite numbers are too low to be detected in blood or tissue smears or by histology. Hemoparasites can also be studied by subinoculation of infectious blood in birds of the same or a known susceptible avian species. Bacteria can usually be identified by blood culture and molecular methods.